PROJECT SUMMARY: This proposal addresses a major clinical challenge that radiologists and other physicians encounter frequently, namely distinguishing active infection from other processes in the human body. Existing clinical tools for detecting infection, including 111In SPECT white blood cell scanning or 18F-FDG PET, target the immune response to bacteria and not the bacteria themselves. Although these modalities can sometimes be useful, they lack the specificity required to distinguish living bacteria from sterile inflammation, cancer, and other highly metabolic tissues. In this proposal, we describe a hyperpolarized (HP) 13C magnetic resonance (MR) imaging technique that identifies metabolic pathways specific to bacteria. HP 13C MR is a powerful technology that has exploded in the recent few years. Put simply, we will employ this modality to develop biomarkers (small molecule byproducts) using HP probes that only bacteria (but not mammalian cells) can make from these precursors. We will show its feasibility without ionizing radiation, and using HP 13C probes that are safe for human use. A fast, high-specificity method of diagnosing infection and its location would revolutionize clinical practice. Here we develop and test 3 novel probes for imaging infection in representative gram positive (S. aureus) and gram-negative (E. coli) bacteria as well as mammalian cells (including cancer cells and activated immune cells) to show specificity to microbial metabolism. We have already shown in preliminary data that HP [2-13C] pyruvate forms significant quantities of [1-13C] acetate when administered to both S. aureus (gram-positive) and E. coli (gram-negative) in vitro. In contrast, we have never observed [1-13C] acetate in mammalian cell lines. In Specific Aim 1, we will optimize a novel MR-compatible 3D culture platform (bioreactor) for studying the bacterial metabolism of hyperpolarized agents in a rigorous fashion. In Specific Aim 2, we will optimize the preparation of the candidate HP probes for dynamic nuclear polarization and use the bioreactor to systematically study its metabolism using the above mentioned cells. Finally, in Specific Aim 3, we will implement optimized MR imaging methods to test our novel HP 13C probe in an in vitro phantom model containing the bacteria and some negative controls, to demonstrate spatial localization of bacteria. Furthermore, HP pyruvic acid has already been used in human phase 1 clinical trials and can be polarized using commercially-available clinical-grade equipment. Clinical polarizers for human use are currently installed in multiple sites throughout the world. Therefore, if successful, the approaches explored in this work will be translatable to clinical imaging at our institution and at other sites.